摘要
目的探讨RNA干扰技术沉默Notch1基因表达对人乳腺癌细胞增殖和凋亡的影响。方法设计并合成靶向Notch1基因的小分子干扰RNA质粒,在转染试剂Sofast介导下转染人乳腺癌细胞株MCF-7,用RT-PCR和Western blot法检测转染前、后Notch1基因的表达,挑选干扰效率最强的一组表达载体;cck8比色法检测分析各组细胞的存活率;流式细胞术检测细胞凋亡比例;Western blot法检测转染各组MCF-7细胞Notch1、NF-κB及Caspase-3蛋白表达。结果 Notch1-shRNA能有效封闭Notch1基因的表达,Notch1基因和蛋白表达水平明显降低(P<0.05);Notch1-shRNA能明显抑制细胞增殖(P<0.05);转染48h后细胞凋亡比例增加(P<0.01)。NF-κB蛋白水平表达降低,Caspase-3蛋白表达水平增高。结论利用RNA干扰技术沉默Notch1基因的表达可以明显抑制MCF-7细胞的增殖,促进MCF-7细胞凋亡,其机制可能通过NF-κB信号通路调节相关凋亡蛋白的表达,进而影响细胞的凋亡和增殖,靶向Notch1的RNA干扰技术在乳腺癌的基因治疗中具有一定的研究价值。
Purpose To explore the proliferation and apoptosis of human breast cancer MCF-7 cell by applying RNA interference tech- nique to silence Notchl gene. Methods Four small fragments of siRNA and one negative control were sequenced and cloned into the vector. The recombinant plasmids were then transfected into breast cancer cell line MCF-7 by Sofast transfection assay. The transcription and translation level of Notehl expression were detected by RT-PCR and Western blot. Meanwhile, the proliferation and apoptosis of transfected tumor cells were determined by cck8 and flow-cytometry assay. The protein expressions of NF-KB and Caspase-3 were detec- ted by Western Blot. Results The recombinant plasmids containing Notchl shRNA were successfully constructed. Notchl expression was significantly knocked down after shRNA plasmid transfection（ P 〈 O. 05 ）. Notchl shRNA visibly inhibited the proliferation of breast cancer cell（P 〈 O. 05 ）and the apoptosis rate increased after 48 h transfection（P 〈 0. O1 ）. The expression of NF-KB decreased but the expression of Caspase-3 increased significantly. Conclusions Notch gene silenced by shRNA can inhibit the proliferation and induce the apoptosis of human breast cancer cell line MCF-7 which may be regulated though NF-KB pathway, and the process might serve as a potential approach for breast cancer gene therapy.
出处
《临床与实验病理学杂志》
CAS
CSCD
北大核心
2012年第1期11-14,共4页
Chinese Journal of Clinical and Experimental Pathology
基金
国家自然科学基金资助项目(30870968)
教育部高等学校博士学科点专项科研基金(200801610001)
